Rapid screening of protein–protein interaction inhibitors using the protease exclusion assay

We have previously developed a sensitive and modular homogenous biosensor system using peptides to detect target ligands. By transposing the basic mechanistic principle of the nuclease protection assay into this biosensor framework, we have developed the protease exclusion (PE) assay which can discern antagonists of protein–protein interactions in a rapid, single-step format. We demonstrate the concept with multiple protein–peptide pairs and validate the method by successfully screening a small molecule library for compounds capable of inhibiting the therapeutically relevant p53–Mdm2 interaction. The Protease Exclusion method adds to the compendium of assays available for rapid analyte detection and is particularly suited for drug screening applications. •A novel homogenous biosensing assay capable of rapid and sensitive detection of peptide–protein interactions.•Readily configurable for both fluorescent and enzyme-linked signal read-out.•Validated for use in screening of a drug library for novel inhibitors of a therapeutically relevant protein–protein interaction.